Method for constructing viral nucleic acids in a cell-free manner

Abstract

The present invention relates to a method for constructing viral nucleic acids in a cell-free manner. In essence, the cell-free method entails the immobilization of a fragment of a double-stranded DNA sequence on a solid support and the assembly of the remaining fragments of the double-stranded DNA sequence onto the immobilized fragment. If the viral nucleic acid is derived from an RNA virus, the instant method further comprises the step of in vitro transcription of the assembled double-stranded DNA sequence to yield an RNA viral nucleic acid.

1 . A method for constructing a viral nucleic acid in a cell-free manner, comprising the steps of: 
 (a) immobilizing a presynthesized first fragment of a double-stranded DNA sequence, which corresponds to a viral nucleic acid sequence, directly on a solid support;    (b) treating said first fragment and a second fragment of said double-stranded DNA sequence with an enzyme having 3′-5′ exonuclease activity, which provides said first fragment and said second fragment each with 5′-extending single-stranded end of defined sequence and length, wherein said single-stranded ends of first and second fragments are complementary to each other;    (c) assembling by hybridization and ligation of said first fragment with said second fragment of said double-stranded DNA sequence ; and    (d) treating and assembling said second fragment and a subsequent fragment according to steps (b) and (c); and    (e) repeating step (d) with subsequent fragments until the double-stranded DNA sequence is fully assembled.    
     
     
         2 . The method according to  claim 1 , wherein said enzyme is T4 DNA polymerase.  
     
     
         3 . The method according to  claim 1 , wherein said solid support is a streptavidin-coated solid support and said first fragment of the double-stranded DNA sequence is biotinylated.  
     
     
         4 . The method according to  claim 1 , wherein said first fragment immobilized on the solid support corresponds to the 3′ portion of said viral nucleic acid.  
     
     
         5 . The method according to  claim 1 , wherein said viral nucleic acid is native to an RNA plant virus.  
     
     
         6 . The method according to  claim 9 , wherein said viral nucleic acid is native to a single-stranded, plus sense RNA plant virus.  
     
     
         7 . The method according to  claim 1 , wherein said viral nucleic acid is native to an animal virus.  
     
     
         8 . The method according to  claim 1 , wherein said viral nucleic acid contains one or more sequences non-native to said viral nucleic acid.  
     
     
         9 . The method according to  claim 1 , wherein said viral nucleic acid contains one or more non-native promoters.  
     
     
         10 . The method according to  claim 9 , wherein said viral nucleic acid contains a non-native sequence fused with a native sequence encoding a coat protein or fragments thereof.  
     
     
         11 . The method according to  claim 12 , wherein said non-native sequence encodes a product selected from the group consisting of enzymes, antibodies, hormones, pharmaceuticals, vaccines, pigments, and antimicrobial polypeptides.  
     
     
         12 . The method according to  claim 12 , wherein said non-native sequence is a regulatory sequence.  
     
     
         13 . The method according to  claim 1 , wherein said method is used for high throughput construction of viral nucleic acids.